基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒  

A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71

作　　者：田梓佚 徐牧岑 胡顺铭 张楚妍 陈琛 李永新[1] TIAN Zi-Yi;XU Mu-Cen;HU Shun-Ming;ZHANG Chu-Yan;CHEN Chen;LI Yong-Xin(West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西公共卫生学院/四川大学华西第四医院,成都610041

出　　处：《分析化学》2023年第1期93-101,共9页Chinese Journal of Analytical Chemistry

基　　金：成都市科技局项目(No.2022-YF05-00296-SN);四川大学大学生创新创业训练计划项目(No.20221291L)资助。

摘　　要：以肠道病毒71型(EV71)为检测对象,建立了基于催化发夹组装(CHA)和荧光共振能量转移(FRET)的生物传感器。设计了检测EV71 VP1基因的CHA信号放大策略,该体系由一对发夹探针(H1、H2)构成,H1和H2分别标记了荧光基团Cy3和Cy5。当体系中有VP1基因时,可引发H1和H2催化自组装反应,从而使得Cy3和Cy5相互靠近并发生FRET,导致Cy3荧光信号降低,Cy5信号增强;当体系中没有VP1基因时,H1和H2则稳定存在于反应体系中,不发生FRET,只检测到Cy3的荧光信号。优化了缓冲液浓度、H1与H2的比例、反应温度和反应时间。在最优条件下,所构建的传感器的Cy5与Cy3的荧光强度比值与EV71 VP1基因浓度在0.5~20 nmol/L范围内呈良好的线性关系,检出限为73 pmol/L(3σ)。咽拭子样品的加标回收率为99.6%~103.1%,相对标准偏差在0.3%~1.7%之间。本方法具有较好的特异性及抗干扰能力,在EV71监测和手足口病早期诊断方面具有良好的应用前景。A biosensor based on catalytic hairpin assembly(CHA)and fluorescence resonance energy transfer(FRET)was established for detection of enterovirus 71(EV71).A CHA signal amplification strategy was designed to detect the VP1 gene of EV71,which was consisted of a pair of hairpin probes(H1 and H2).The fluorophores Cy3 and Cy5 were labeled on H1 and H2,respectively.When the VP1 gene was present,the catalytic assembly reaction of H1 and H2 was triggered,Cy3 and Cy5 got closer and FRET occurred,leading to a reduced Cy3 signal and an enhanced Cy5 signal.In the absence of VP1 gene,H1 and H2 existed stably in the reaction system and could not generate FRET.Experimental conditions including reaction buffer concentration,the ratio of H1 to H2,reaction temperature and reaction time were optimized.Under the optimized conditions,the fluorescence intensity ratio of Cy5 to Cy3 showed a good linear relationship with EV71 VP1 gene concentration in the range of 0.5-20 nmol/L,and the detection limit was 73 pmol/L(3σ).The spiked recoveries of throat swab samples ranged from 99.6%to 103.1%with the relative standard deviations of 0.3%to 1.7%,showing good specificity and anti-interference ability.The proposed sensor had a good application prospect in EV71 virus monitoring and early diagnosis of hand,foot and mouth disease.

关 键 词：肠道病毒71型 催化发夹组装 荧光共振能量转移 生物传感器 手足口病 

分 类 号：O657.3[理学—分析化学] R446.5[理学—化学]